KR20150126350A - Pharmaceutical formulation containing glycosaminoglycan - Google Patents

Abstract

A pharmaceutical formulation comprising glycosaminoglycan is provided. In this pharmaceutical formulation, the glycosaminoglycan layer surrounding the drug layer is used to improve the therapeutic efficiency of bowel disease. According to one embodiment, the pellet comprises a core, a drug layer surrounding the core, a glycosaminoglycan layer surrounding the drug layer, a separation layer surrounding the glycosaminoglycan layer, Includes an enteric coating layer. Accordingly, the weight ratio of the core, the drug layer, the glycosaminoglycan layer, the separation layer, and the enteric coating layer is 100: 137-141: 25-29: 9-13: 60-64, Is 100: 139: 27: 11: 62.

Description

[0001] PHARMACEUTICAL FORMULATION CONTAINING GLYCOSAMINOGLYCAN [0002] PHARMACEUTICAL FORMULATION CONTAINING GLYCOSAMINOGLYCAN [0003]

This application claims priority to International Application No. PCT / US2013 / 031817, filed March 15, 2013, which is incorporated herein by reference.

The present invention relates to pharmaceutical preparations. More specifically, the present invention relates to pharmaceutical preparations comprising glycosaminoglycans.

Enteric coatings have been developed over a long period of time to improve the therapeutic efficacy of intestinal diseases, thereby reducing the dose of drug used. However, there is still a need to improve the therapeutic efficacy of bowel disease in order to further reduce the dose of drug used.

Mesalamin, also known as mesalazine or 5-aminosalicylic acid (5-ASA), is an anti-inflammatory drug used to treat digestive tract inflammation, ulcerative colitis, and mild to severe Crohn's disease. Mesalamin is an intestinally specific aminosalicylate drug that works locally in the digestive tract and has a significant side effect due to its marked action there. As a derivative of salicylic acid, it is believed that mesalamine is an antioxidant that captures free radicals that potentially damage the by-products of biological metabolism. N-acetyl-5-ASA is a metabolite of 5-ASA. Absorbed 5-ASA is rapidly acetylated by the liver through the mucosal wall of the digestive tract. It is excreted primarily by the kidney as N-acetyl-5-ASA.

U.S. Patent No. 4,980,173 discloses that a pharmaceutical composition comprising 5-aminosalicylic acid or a pharmaceutically acceptable salt or ester thereof as an active ingredient can be used to treat ulcerative colitis or Crohn's disease by oral administration. Certain sustained release tablet formulations and their manufacture have been disclosed. The disadvantage of this patent is that the drug is naturally distributed in the intestine and not dispersed in a particular site, so that a larger dose is needed to achieve better treatment outcomes, a lower drug dose attached to the inflamed site, .

U.S. Patent No. 5541170 discloses a pharmaceutical composition comprising a pharmacologically active ingredient coated with an anionic polymer that is insoluble in the gastric juice of less than pH 7 and insoluble in the colloid solution and which is soluble in the colloid solution in an amount sufficient to remain intact until the oral dosage form reaches the colon ≪ / RTI > or a capsule or tablet containing the active ingredient. This invention specifically releases the drug in an environment below pH 7, but does not target the target to increase the drug concentration relative to the lesion. This disadvantage is the same as described above.

U.S. Patent No. 5541171 also discloses a solid dosage form similar to U.S. Pat. No. 5541170, which adds more limiting elements to the previous patent.

U.S. Patent No. 6551620 discloses an oral pharmaceutical pellet preparation for the treatment of digestive tract comprising a core and an enteric coating, wherein the core comprises an amino salicylic acid or a pharmaceutically acceptable salt or derivative thereof as a pharmaceutically active compound . The disadvantage is still the same as described above.

United States Patent No. 6773720 discloses a controlled release oral pharmaceutical composition comprising 5-amino salicylic acid as an active ingredient. Said composition comprising: (a) an internal lipophilic matrix consisting of a material having a melting point of less than 90 DEG C, the active ingredient being at least partially inglobated; (b) an external hydrophilic matrix in which the lipophilic matrix is dispersed; And (c) optionally, other additives. The disadvantage is still the same as described above.

U.S. Patent No. 6893662 discloses a pharmaceutical composition in solid unit dosage form for oral administration to a human or lower animal. The pharmaceutical composition comprises (a) a safe and effective dose of the therapeutically active substance; (b) an inner coating layer selected from the group consisting of poly (methacrylic acid, methyl methacrylate) 1: 2, poly (methacrylic acid, methyl methacrylate) 1: 1, and mixtures thereof; And (c) an outer coating layer comprised of an enteric polymer or film coating material. However, the disadvantage is still the same as described above.

U.S. Pat. No. 6,046,179 discloses a method of treating inflammatory bowel disease in a patient suffering from inflammatory bowel disease comprising (a) a therapeutic dose of N-acetyl-glucosamine, (b) a pharmacologically acceptable carrier suitable for administration to the patient's colon ≪ / RTI > The disadvantage of this patent is the lack of drugs other than N-acetyl-glucosamine; Thus, there is a lack of supplementation between the other drugs, such as N-acetyl-glucosamine and mesalamine.

Since the compositions of the aforementioned patents must be used under a regular dose of each drug or N-acetyl-glucosamine, these compositions can not reduce the general therapeutic dose of each drug.

Glycosaminoglycan can be obtained from a variety of sources, such as chicken bran, bronchi, umbilical cord, skin, joint fluid and certain bacteria such as streptococcal species. Most glycosaminoglycans consist of repeating sugars, such as N-acetylglucosamine, N-acetylglucuronic acid and / or N-acetylgalactosamine, which are known as non-sulfated glycosaminoglycans. do. When such glycosaminoglycans include sulfur groups, they are known as sulfated glycosaminoglycans. Examples of glycosaminoglycans include hyaluronic acid (consisting of repeating units of N-acetylglucosamine and glucuronic acid), chondroitin sulfate, dermatan sulfate, keratan sulfate and heparin, all of which are N-acetylglucosamine or Amino sugar, N-acetal galactosamine. Glycosaminoglycan also appears in proteoglycans, a structure comprising several glycosaminoglycan chains linked to a polypeptide or protein core.

Accordingly, in one embodiment, the present invention relates to a pharmaceutical preparation comprising at least pellets for improving the therapeutic efficiency of bowel disease.

According to another embodiment, the pellet comprises a core, a drug layer surrounding the core, a glycosaminoglycan layer surrounding the drug layer, a separation layer surrounding the glycosaminoglycan layer, And an encapsulating enteric coating layer.

Accordingly, the weight ratio of the core, the drug layer, the glycosaminoglycan layer, the separation layer, and the enteric coating layer is 100: 137-141: 25-29: 9-13: 60-64, : 139: 27: 11: 62.

According to another embodiment, the pellet comprises a core, a drug layer surrounding the core, a glycosaminoglycan layer surrounding the drug layer, and an enteric coating layer surrounding the glycosaminoglycan layer.

Accordingly, the weight ratio of the core, the drug layer, the glycosaminoglycan layer and the enteric coating layer is 100: 137-141: 25-29: 60-64, preferably 100: 139: 27: 62 to be.

In one embodiment, the core comprises a pharmaceutically acceptable inert material such as cellulose, starch, sugar, or silicon oxide, preferably microcrystalline cellulose.

In another embodiment, the drug layer comprises a drug for the treatment of bowel disease.

In another embodiment, the drug layer is selected from the group consisting of mesalamine, a laxative agent, a glucocorticoid, an antimicrobial agent, an immunosuppressant, a chemotherapeutic agent, an anti-cancer agent, a peptide, a protein, a cardiovascular agent, a psychotropic agent, , Steroids, non-steroidal anti-inflammatory drugs (NSAIDs), antibiotics, anti-inflammatory agents, or derivatives thereof.

In another embodiment, the drug layer further comprises a binder such as hydroxypropylmethylcellulose, hydroxypropylcellulose, or polyvinylpyrrolidone.

In another embodiment, the glycosaminoglycan layer includes, for example, hyaluronic acid or salt thereof, chondroitin sulfate, heparin sulfate, heparin, keratan sulfate, or dermatan sulfate.

In another embodiment, the separation layer comprises a hydrophobic polymer that is soluble at least above pH 5.5. The hydrophobic polymer may be, for example, 1: 1 poly (methacrylic acid-co-ethylacrylate), 1: 1 poly (methacrylic acid-co-methyl methacrylate) Methacrylate-co-methacrylic acid) 7: 3: 1, cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, methylhydroxypropyl-cellulose phthalate, ethylhydroxycellulose phthalate, polyvinyl acetate phthalate, polyvinyl Maleic anhydride copolymers, styrene-maleic acid monoester copolymers, methyl acrylate-methacrylic acid copolymers, and methacrylate-methacrylic acid-octylacrylate copolymers.

In another embodiment, the separating layer or enteric coating layer comprises an enteric polymer or a pH-resistive polymer that can be dissolved at a pH of at least 6.8. The enteric polymer may be selected from, for example, cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, ethylhydroxycellulose phthalate, polyvinylacetate phthalate, polyvinyl butyrate acetate, vinyl acetate-maleic anhydride copolymer, styrene- Monoester copolymers, methyl acrylate-methacrylic acid copolymers, methacrylate-methacrylic acid-octyl acrylate copolymers, or any combination thereof.

In another embodiment, the pellet is present in a capsule or tablet.

The foregoing embodiments are provided to provide a simplified summary of the present disclosure in order to provide a basic understanding of the present invention. This summary is not an extensive overview of the present specification, and does not define or describe in detail the essentials / key elements of the present invention. Its full purpose is to provide some technical ideas described herein in a simplified form as a preamble to a more detailed description to be provided later. Many of the accompanying features will be more readily appreciated and understood with reference to the following detailed description together with the drawings.

The present invention provides pharmaceutical preparations containing at least pellets for improving the therapeutic efficiency of intestinal diseases.

1A is a structural view of a pellet according to an embodiment of the present invention.
1B is a structural view of a pellet according to another embodiment of the present invention.
Figure 2 shows the affinity of HA for fluorescence indices in normal and damaged colon tissues (* p < 0.05).
Figure 3 shows the colon tissue biopsy time, mesalamine concentration profile after administration of Colasa® (brand name enema with 20 mg mesalamine in 1 ml solution) or HA-mesalamine.
Figures 4A and 4B are dissolution profiles of pellets dissolved in pH 6.8 and pH 4.5 buffer solutions.

In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are shown in schematic form in order to simplify the drawings.

Definition of Terms

The term "subject" as used herein includes an individual (patient) in need of treatment of a drug (e.g., mesalamine) and an individual that does not require medication (e.g., normal healthy volunteers).

A "therapeutically effective amount" or "effective amount" of a drug (eg, mesalamine) is the amount necessary to achieve the desired pharmacological effect or therapeutic improvement without undue side effects. The effective amount of the drug (e. G. Mesalamine) will be selected by those skilled in the art based on the particular patient and the name of the subject. Effective amount "or" therapeutically effective amount "refers to an amount of a compound (e.g., a pharmaceutically acceptable salt, solvate, prodrug, It can be understood that it may vary depending on the individual. Moreover, a person skilled in the art can clearly understand "quantity" or "dose" depending on the packaging insert or label of the registered drug in the drug store approved by the drug management authority.

"Treatment" or "treatment" is intended to prevent a disorder or disease in an individual who tends to have a disorder or disease but is not yet diagnosed with the disorder or disease; Inhibiting the disorder or disease by inhibiting the progress of the disorder or disease, alleviating the disorder or disease, inducing recovery of the disorder or disease, alleviating the condition caused by the disorder or disease, &Lt; / RTI &gt; such as reducing the symptoms of &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt;

Introduce

The present invention is based on the inventor's test results that the amount of hyaluronic acid (HA) that binds to the inflammatory surface is higher in the non-inflammatory area of the colon tissue. Moreover, the results indicate that when HA is mixed with the drug, HA can act as a transport medium to deliver drugs suitable for the treatment of inflammation and / or allergy and / or damage such as mesalamine. Thus, the dose of the drug can be reduced compared to the usual dose, and the therapeutic effect is also greatly improved due to the drug particularly adhering to the place where treatment is required. Therefore, the present invention further provides the preparation of the present invention based on the above-mentioned results.

Pellet  Containing pharmaceutical preparations

It is therefore a feature of the present invention to provide a pharmaceutical formulation comprising at least pellets in order to improve the therapeutic efficiency of intestinal diseases. The pellet is designed to release the drug at a location farther away than that which is released by the immediate release dosage form in the gastrointestinal tract and / Controlled release formulation (also known as sustained release formulation).

The structure of each pellet in this pharmaceutical preparation is shown in Fig. Pharmaceutical formulations include capsules, tablets, or other types of formulations that require at least a pellet. That is, the pellets can be encapsulated in capsules or extruded into tablets.

1A is a structural view of a pellet according to an embodiment of the present invention. 1A, a pellet 100a includes a core 110, a drug layer 120, a glycosaminoglycan layer 130, a separation layer 140, and an enteric coating layer 150 in this order from the inside to the outside . The weight ratios of the core 110, the drug layer 120, the glycosaminoglycan layer 130, the separation layer 140, and the enteric coating layer 150 are shown in Table 1.

The weight ratio of each part in the pellet (100a) ingredient Weight ratio The core 110, 100 The drug layer 120 137-141 The glycosaminoglycan layer (130) 25-29 The separation layer 140, 9-13 The enteric coating layer (150) 60-64

The core 110 is used as a coating seed to facilitate coating of subsequent layers. Therefore, the diameter of the core 110 is about 500-800 μm, for example, 700 μm. The composition of core 110 may be any pharmaceutically acceptable inert material, such as, for example, cellulose, starch, sugar, or silicon dioxide. The cellulose may be, for example, methyl cellulose, hydroxypropyl methylcellulose, sodium carbomethyl cellulose, or microcrystalline cellulose. The starch may be, for example, maize starch, wheat starch, rice starch, potato starch or corn starch, or may be derived therefrom. The saccharide can be, for example, maltose, lactose, fructose, galactose, trehalose, sucrose, mannitol or sorbitol.

The drug layer 120 contains a drug suitable for treating bowel disease. For example, for treatment of enteritis, the drug may be an antibiotic or an antiseptic. For the treatment of gastric ulcers, the drug may be a coagulant, an antibiotic, an antacid, an H2 blocker, a potassium hydrogen ion pump blocker (proton pump inhibitor, PPI), a cytoprotective agent or a mucosal protective agent. For the treatment of inflammatory bowel disease (IBD), the drug may be steroids, immunosuppressants, antibiotics, 5-ASA (5-aminosalicylic acid) and derivatives, or anti-inflammatory agents. Thus, for example, the drug may be any of the above. Preferably, the drug in the drug layer 120 comprises a positively charged functional group.

The drug layer 120 may further comprise a binder for the force with which the drug binds to the core 110. The binder may be, for example, hydroxypropylmethylcellulose, hydroxypropylcellulose, or polyvinylpyrrolidone.

The glycosaminoglycan layer 120 mainly contains glycosaminoglycan used for crosslinking the drug in the drug layer 120 and the intestinal mucosa in the gastrointestinal tract. Glycosaminoglycan can be hyaluronic acid, chondroitin sulfate, heparin sulfate, heparin, keratan sulfate or dermatan sulfate.

According to this embodiment, glycosaminoglycan is hyaluronic acid. Hyaluronic acid (hereinafter abbreviated as 'HA') is an anionic, non-sulfurized, glycosaminoglycan widely distributed throughout the bond, epithelium and nervous tissue. Therefore, HA has high affinity to living tissue such as mucous membranes. Since HA contains negatively charged carboxylate groups, HA can interact with positively charged functional groups. Thus, if the drug in the drug layer 120 has a positively charged functional group, the HA can interact with the drug to fix the drug to the intestinal mucosa to concentrate the drug at a particular site, such as a damaged area; Thus reducing the loss of administered drug, improving the therapeutic effect on bowel disease and the efficacy and efficacy of the drug. This phenomenon can also be understood by the results of a prototype experiment of the present invention, showing that it is more firmly attached to the damaged region than the normal region.

The separation layer 140 is used to protect the glycosaminoglycan layer 130 from contact with water during the subsequent pelletization step. When the glycosaminoglycan in the glycosaminoglycan layer 130 is HA, since the HA will easily form an HA film after contact with water, there is a problem that pellets aggregate and interfere with the coating. Thus, the material used for the separation layer 140 may be a hydrophobic polymer that is soluble at least at pH 5.5.

Wherein the hydrophobic polymer is selected from the group consisting of poly (methacrylic acid-co-ethylacrylate) 1: 1, poly (methacrylic acid-co-methyl methacrylate) 1: 1, poly (methyl acrylate- Cellulose acetate phthalate, cellulose acetate succinate, methyl cellulose phthalate, methylhydroxypropyl-cellulose phthalate, ethylhydroxy cellulose phthalate, polyvinylacetate phthalate, polyvinyl butyrate acetate, vinyl acetate Maleic anhydride copolymers, styrene-maleic acid monoester copolymers, methyl acrylate-methacrylic acid copolymers, or methacrylate-methacrylic acid-octylacrylate copolymers. These hydrophobic polymers may be used singly or in combination, and may be used together with polymers other than those mentioned above.

The enteric coating layer 150 mainly contains an enteric polymer that can be preferentially dissolved in the small intestine, the large intestine, or both, of an environment less acidic than the acidic environment above. It is expected that any anionic polymer that exhibits a pH-dependent solubility profile may be used as a functional coating in the practice of the present invention. According to this embodiment, the enteric polymer can be dissolved at least at pH 6.8. The enteric polymer may be selected from the group consisting of cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, ethylhydroxycellulose phthalate, polyvinylacetate phthalate, polyvinyl butyrate acetate, vinyl acetate-maleic anhydride copolymer, styrene-maleic acid monoester copolymer , Methyl acrylate-methacrylic acid copolymer, methacrylate-methacrylic acid-octyl acrylate copolymer, and the like. These enteric polymers may be used singly or in combination, or may be used together with polymers other than those mentioned above.

1B is a structural view of a pellet according to another embodiment of the present invention. In FIG. 1B, the separation layer 140 of the pellet 100a in FIG. 1A is omitted in forming the structure of the pellet 100b.

In pharmaceutical preparations Pellet  How to make

Another feature of the present invention is to provide a method for making pellets filled in capsules or tablets. Each layer of pellets 100a or 100b shown in FIG. 1A or 1B is coated by a fluid bed system (Huttlin, Germany). The coating solution for each layer of the pellets 100a or 100b will be described later.

For coating of drug layer 120, a drug coating solution is prepared by the following steps. First, at least one of the binders described above is added to water and stirred to form a clear solution having a viscosity of 4.8 to 7.2 cP (centipoise). Then, antistatic agent and drug are sequentially added to the transparent solution of the binder and stirred to form a uniform drug coating solution. Anti-static agents such as talc (i.e., hydrated magnesium silicate) are used to prevent agglomeration of the pellets.

For the coating of the glycosaminoglycan layer 130, a glycosaminoglycan coating solution is prepared by the following steps. First, the binder is added to ethanol (99.9%) and stirred to form a transparent solution. The glycosaminoglycan is then added to the clear solution of the binder and stirred to form a uniform glycosaminoglycan coating suspension. The binder may be hydroxypropyl cellulose (e.g., HPC-L).

For the coating of the separating layer 140, the separating coating solution is prepared by the following steps. The hydrophobic polymer is added to ethanol (95%) and stirred to form a clear solution. Triethyl citrate and talc are then added to the clear solution of the hydrophobic polymer and stirred to form a uniform separation coating solution.

For the coating of the enteric coating layer 150, an enteric coating solution is prepared by the following steps. At least an enteric polymer, an emulsion inclusion, glyceryl monostearate, triethyl citrate and polysorbate 80 (PlasACRYL-T20) and water are mixed to form a homogeneous solution. The solution is filtered through a 60 mesh sieve.

Example  1: Attachment of HA in colon tissue

step:

(I) 0.25 g of a high molecular weight sodium hyaluronate powder (abbreviated as 'HHA', MW: 2 MDA; Freda) and 0.25 g of a low molecular weight sodium hyaluronate powder (abbreviated as 'LHA', MW: 350 kDa; Freda) Buffer (phosphate buffered saline) to form a 0.5% solution, followed by stirring for 6 hours until the powder was completely dissolved. 0.05 g of LHA powder and 0.2 g of HHA powder (ratio 2: 8, average MW of 1 MDa; hereinafter referred to as "medium molecular weight sodium hyaluronate powder and abbreviated as" MHA ") were added to a 50 ml PBS buffer, Lt; / RTI &gt; for 6 hours.

(II) Fluorescent HA (abbreviated as 'HA-f') was prepared by the following steps;

(1) MES buffer solution: 0.39 g of MES free acid (2- (N-morpholino) ethanesulfonic acid, Calbiochem) was dissolved in 100 ml of dd (secondary distilled) water.

(2) Solution A: 65 mg of a fluorocinamine powder (isomer I, Fluka) was dissolved in 9 ml of a 95% ethanol solution, and the mixture was stirred for 10 minutes in a state where the light was blocked.

(3) Solution B: 359 g of EDC powder [N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride, Sigma] was dissolved in 9 ml of MES buffer and stirred for 10 minutes.

(4) Solution C: 216 mg of NHS powder (N-hydroxysuccinimide, Sigma) was dissolved in 9 ml of MES buffer, followed by stirring for 10 minutes.

(5) 3 ml of the solution A was slowly added dropwise to 50 ml of a 0.5% HA solution, and the mixture was stirred for 10 minutes while the light was blocked.

(6) 3 ml of the solution B and 5 ml of the solution C were each added dropwise to the solution of the step (5), and then the mixture was stirred for 10 minutes in the state that the light was blocked.

(7) A 0.02 M MES buffer solution was added to the solution of step (6), slowly added until the volume reached 100 ml, and then the mixture was stirred at room temperature for 24 hours in the state of blocking light.

(8) After the reaction, the product was poured into a dialysis tube (MW: 12000 to 14000) in a 5 L secondary distilled water as a dialysis solution, and then the dialysis solution was changed every 12 hours until the dialysis solution had no fluorescence, Lt; RTI ID = 0.0 &gt; 4 C &lt; / RTI &gt; for 5 days.

(9) After dialysis, the liquid was dispensed into a 50 mL plastic centrifuge tube, stored overnight in a -20 ° C refrigerator, and dried in a freeze-drying machine with the light blocked.

(10) The dried HA-f powder was stored in a -20 ° C freezer.

(11) 50 mg of HA-f powder was slowly added to 10 ml of PBS buffer and stirred for 6 hours until the powder was completely dissolved.

(III) The colonic tissues of SD-rats (Sprague-Dawley Rat) aged 7 to 8 weeks were cut with a scalpel, washed with PBS buffer, cut into 3-4 cm length and immersed in PBS buffer.

(IV) toothbrush 20 times and immersed in PBS buffer to prepare damaged colon tissues.

(V) Normal and injured colon tissues were placed in a 12-well plate, 1 ml of 0.5% HA-f solution was added to each well, and the mixture was stirred at room temperature for 2 hours. The excess HA-f solution was suctioned after 2 hours by a tip, then immersed in PBS buffer for 10 minutes, and the PBS buffer was removed three times.

(VI) The washed colon tissue was placed in a 12-well plate, with the tissues facing up, and placed in the dock of IVIS (in vivo imaging system, XENOGEN). The default parameters were set as GFP (Green Fluorescent Protein), this was 465 nm, the emission was 500 nm, and the image was captured in software.

(VII) All values in the table are expressed as an average of n observations. Histological index was analyzed by Student's t-test.

result:

Figure 2 shows the affinity of HA for normal and damaged colon tissues by fluorescence indices (* p < G.05). In Fig. 2, the fluorescence index of normal colon tissue was defined as 1. Other colon tissue tests were graded by fluorescence index of normal colon tissue. The results showed that the adherence of HA with the same average molecular weight was significantly higher in injured colon tissues than in normal colon tissues (P < 0.01). Contrasting the different loading of HA with three average molecular weights (i.e., HHA, MHA, and LHA) in damaged colon tissues, the fluorescence index of attachment of 350 KDa HA (i.e., LHA) And the other two HA with 1 MDa (i.e., HHA and MHA).

Example  2: different Mesalamine  Formulation In the body cavity  After input Mesalamine  Comparative analysis of colon tissue concentration

step:

(I) Experimental animals:

8-week-old male SPF-grade SD (Sprague-Dawley) rats (280-330 g) were obtained from BioLASCO Taiwan Co., Ltd.

(Ⅱ) Experimental drug:

(A) Colasa® enema (20 mg / ml, United Biomedical, inc. Asia), and

(B) 0.25% (w / w) HA mixture (abbreviated as 'HA-M') (8: 2 = 2000 KDa HA: 350 KDa HA) in PBS (pH 7.4) containing 5 mg / mL mesalamine.

(Ⅲ) Intra-body injection of experimental drug:

After lightly anesthetizing with Jorretil 50, rat abdominal incision was performed with surgical scissors and the colon was confirmed. Two sections of the colon (2 cm each) were tied with cotton yarn and 0.5 ml of the experimental drug was injected into the body cavity of the isolated colon fragment. After 0.5, 1, 1.5 or 2 hours, the rats were sacrificed and the colon fragments removed. Three rats were used at each time point in the cortical infusion.

(IV) Preparation of sample:

Tissue biopsies were washed with PBS solution to remove surface contamination, weighed and immediately frozen in liquid nitrogen and stored at -80 ° C until use. Biopsy was crushed and added to a solution of 50mM KH ₂ PO ₄ (pH 7.4). The tissue cells were ultrasonically stirred for 10 seconds using a microprobe inserted in the suspension, and then ultrasonic agitation was stopped at 25 W for 20 seconds for a total of 10 minutes. After mixing with vortex, the sample was allowed to stand at room temperature for 30 minutes to allow protein precipitation to occur, followed by centrifugation at 13000 g for 30 minutes.

(Ⅴ) Analysis of mesalamine concentrations in colon biopsy:

Mesalamin was measured by ultra high performance liquid chromatography (UPLC). The method is authorized. A Waters (UK) ACQUITY system and a fluorescence detector (excitation 315 nm, emission 430 nm) were used and the data was analyzed using Empower 2. The ACQUITY column (C18, 100x2.1 mm inner diameter, 1.7 mu m particle) purchased from Waters, UK was protected by a Van guard column (C18, 5x2.1 mm inner diameter, 1.7 mu m particle, Waters) . The mobile phase consisted of 0.1 M acetic acid and acetonitrile (850: 150) with triethylamine at pH 4.3. The flow rate was 0.2 mL / min, the resulting pressure was 5400 psi and the analysis was performed at 40 &lt; 0 &gt; C. The injection amount was 5 μl. Samples were derived using propionic acid anhydride to improve the fluorescence properties of mesalamine. Triethylamine was used as an ion pair reagent to improve peak symmetry. The UPLC method of mesalamine analysis was certified to measure mesalamine over a nominal linear range of 10-1000 ng / ml. The linear correlation coefficient (R2) of the method used in this experiment is 1.00.

result:

Figure 3 shows the colon tissue biopsy time, mesalamine concentration profile after injection of Colasar ® (brand name enema with 20 mg of mesalamine in 1 ml solution) or HA-mesalamine (abbreviated as HA-M). 3, the (median) concentration of mesalamine in the colon biopsy after injection of Cola ® reached a peak after one hour of injection. Thereafter, the concentration of mesalamine rapidly dropped. Conversely, HA-M continuously released mesalamine for 2 hours, and the concentration of mesalamine was higher than that of Corsa® after 2 hours of injection.

The results of this example demonstrate that HA-M keeps the release of mesalamine much longer than the commercially available mesalamine enema (Colasar®). However, HA-M contains only 1/4 of the mesalamine concentration in Colasar®. The results of the data showed that HA-M had almost the same efficacy in ameliorating IBD inflammation in artificially induced rats in rats, although only a quarter of the usually therapeutically effective dose was used in HA-M.

Example  3: Pellet  Dissolution experiment

In this example, the pellets were produced according to the provided manufacturing method. The materials used in each layer are shown in Table 2 below.

layer Component (trade name) Weight (mg) ^* Diameter or ^* Thickness (㎛) core Microcrystalline Cellulose (Cellets 500) 160.0 652
Drug layer
Talc 8.0
900
Hydroxypropyl methylcellulose (HPMC 606) 15.0 Mesalamin (drug) 200.0
Glycosaminoglycan layer Sodium hyaluronate 31.25
911 Hydroxypropylcellulose (Grade L) 11.75
Separation layer
Poly (methacrylic acid-co-methyl methacrylate) 1: 1 (Eudragit L 100) 11.0
935
Talc 6.0 Triethyl citrate 1.0
Enteric coating layer
Poly (methyl acrylate-co-methyl methacrylate-co-methacrylic acid) 7: 3: 1 (Eudragit FS30D) 81.0
943
Poly (methacrylic acid-co-ethylacrylate) 1: 1 (Eudragit L30D-55) 9.0 Glyceryl monostearate, triethyl citrate, and polysorbate 80 (PlasACRYL-T20) were added to the above- 9.0

* Measured by scanning electron microscope (SEM)

A dissolution experiment was then performed on the pellet. In this dissolution experiment, 6 capsules were used for each dissolution experiment. The pH values of the buffer solutions used to dissolve the capsules were 4.5 and 6.8, respectively. pH 4.5 buffer solutions were prepared by mixing KH ₂ PO ₄ and H ₃ PO ₄ solutions and pH 6.8 buffer solutions were prepared by mixing KH ₂ PO ₄ , NaOH and KOH solutions. The dissolution experiments were performed at 37.0 ± 0.5 ° C and the paddles were rotated at 100 rpm. An on-line UV detector was set at 330 nm to detect the concentration of dissolved mesalamine. Various amounts of mesalamine were dissolved in a predetermined amount of a pH 4.5 or pH 6.8 buffer solution to provide a standard sample for detecting the concentration of dissolved mesalamine.

Figures 4A and 4B are dissolution profiles of pellets dissolved in pH 6.8 and pH 4.5 buffer solutions. 4A, the dissolution rate of the pellets rapidly increased in 90 minutes. However, in Figure 4b, the pellets were not dissolved in a pH 4.5 buffer solution for a minimum of 2 hours. Thus, it can be seen that the pellets of this example can be dissolved in a pH 6.8 buffer solution, i. E., The intestinal environment, but not in a pH 4.5 buffer solution, i.

Example  4: For colitis DNBS - Induced Pig  In the model The pellet 100a  Therapeutic efficacy

step:

(I) pigs were selected from LY species piglets (Landrace; The age of the experiment was 9 weeks ± 1 week. Nine pigs were divided into three groups: A, B and C.

(II) Reagent:

(a) 40 mg / ml of Stresnil (manufactured by China Chemical & Pharmaceutical Co., Ltd., Taiwan)

(b) 50 mg / ml Zoletil-50 (Virbac Laboratories, France) for the anesthesia of pigs.

(c) Atropine 1 mg / ml (Tai Yu Chemical & Pharmaceutical Co., Ltd., Taiwan)

(d) 150 mg / ml of 2,4-dinitrobenzenesulfonic acid (DNBS) dissolved in 50% ethanol (Sigma-Aldrich Co., USA)

(III) Experimental drug:

Group A: An enteral coated capsule pellet (100a) containing an active dose of 200 mg 5-ASA /

Group B: As a control group, starch

Group C: As a reference drug, Pentasa (trade name) containing 500 mg 5-ASA /

(IV) All pigs were fasted for 2 days. On day 1, the pigs were anesthetized by a gastrointestinal endoscope to examine the condition of the colon.

(V) 40 ml of DNBS (150 mg / mL) was administered rectally and maintained in the colon for 1 hour to induce colitis; The remaining DNBS was then removed and the rectum was washed with 100 ml of distilled water.

(VI) On days 7, 14, 35 and 49, intestinally induced conditions were observed and recorded at the 40, 35, 30, 25, 20, 15, 10, and 5 cm sites from the anus.

(VII) On day 8, the experimental drug was administered twice daily for 28 days according to each group. The daily dosing time was 9.00-11.00 and 16: 30-18: 30.

Blood samples were collected on days 0 (before induction), 7 (after induction), 8 (on the first day of the experimental drug administration), and 12, 14, and 35 days by heparin lithium anticoagulation tube. Tubes were centrifuged at 3000 rpm and analyzed.

(IX) On day 35, tissues with inflammation and normal tissues were obtained by endoscopic biopsy (about 5-10 cm from the anus).

result:

The results of the recovery rates of the different groups during the period are shown in Table 3. From Table 3 it can be seen that group A (HA + 400 mg 5-ASA / day) has a better recovery rate than group B (placebo) and group C (1000 mg 5-ASA / day). This result demonstrates that the effect of the HA + drug can reduce the dose or amount of drug used, which is consistent with the results of Example 2 above. Thus, the formulation of pellet 100a has a better therapeutic effect than a single dose of a normal dose.

* The value of the recovery rate is expressed as mean ± SD.

The concentrations of 5-ASA (i.e., mesalamine) and N-acetyl-5-ASA (metabolites of 5-ASA) in different groups of plasma are shown in Table 4. The lower the concentration of 5-ASA in plasma, the less side effects. In Table 4, the mean concentration of 5-ASA in plasma of group A was lower than the mean concentration of group C, indicating that the dosage form of the HA + drug solved the safety problem.

* Plasma concentrations are expressed as mean ± SD.

Claims (12)

A pharmaceutical formulation comprising at least a pellet,
Wherein the pellet comprises:
A core comprising a pharmaceutically acceptable inert material;
A drug layer surrounding the core and comprising a therapeutically effective amount of a drug for the treatment of bowel disease;
A glycosaminoglycan layer directly surrounding the drug layer and comprising glycosaminoglycan;
A separating layer surrounding the glycosaminoglycan layer and comprising a hydrophobic polymer dissolved at at least pH 5.5; And
And an enteric coating layer surrounding the separating layer and comprising an enteric polymer dissolved at at least pH 6.8. The method according to claim 1,
Wherein the weight ratio of the core, the drug layer, the glycosaminoglycan layer, the separation layer, and the enteric coating layer is 100: 137-141: 25-29: 9-13: 60-64. A pharmaceutical formulation. A pharmaceutical formulation comprising at least a pellet,
Wherein the pellet comprises:
A core comprising a pharmaceutically acceptable inert material;
A drug layer surrounding the core and comprising a therapeutically effective amount of a drug for the treatment of bowel disease;
A glycosaminoglycan layer directly surrounding the drug layer and comprising glycosaminoglycan; And
And an enteric coating layer surrounding the glycosaminoglycan layer and comprising an enteric polymer dissolved at at least pH 6.8. The method of claim 3,
Wherein the weight ratio of the core, the drug layer, the glycosaminoglycan layer, and the enteric coating layer is 100: 137-141: 25-29: 60-64. The method according to claim 1 or 3,
Wherein said pharmaceutically acceptable inert material is cellulose, starch, sugar, or silicon oxide. The method according to claim 1 or 3,
Wherein the drug layer further comprises a binder. The method according to claim 6,
Wherein the binder is hydroxypropylmethylcellulose, hydroxypropylcellulose, or polyvinylpyrrolidone. The method according to claim 1 or 3,
The drug may be selected from the group consisting of mesalamine, a laxative agent, a glucuronide, a glucocorticoid, an antibacterial agent, an immunosuppressant, a chemotherapeutic agent, an anticancer agent, a peptide, a protein, a cardiovascular agent, a psychotropic agent, (NSAID), an antibiotic, an anti-inflammatory agent, and a derivative thereof. The method according to claim 1 or 3,
Wherein the glycosaminoglycan is hyaluronic acid or a salt thereof, chondroitin sulfate, heparin sulfate, heparin, keratan sulfate, or dermatan sulfate. The method according to claim 1 or 3,
Wherein the hydrophobic polymer is selected from the group consisting of poly (methacrylic acid-co-ethylacrylate) 1: 1, poly (methacrylic acid-co-methyl methacrylate) 1: 1, poly (methyl acrylate- Cellulose acetate phthalate, cellulose acetate succinate, methyl cellulose phthalate, methylhydroxypropyl-cellulose phthalate, ethylhydroxy cellulose phthalate, polyvinylacetate phthalate, polyvinyl butyrate acetate, vinyl acetate Maleic anhydride copolymers, styrene-maleic anhydride copolymers, styrene-maleic acid monoester copolymers, methyl acrylate-methacrylic acid copolymers, and methacrylate-methacrylic acid-octyl acrylate copolymers. Lt; / RTI &gt; The method according to claim 1 or 3,
The enteric polymer may be selected from the group consisting of cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, methylhydroxypropyl-cellulose phthalate, ethylhydroxycellulose phthalate, polyvinylacetate phthalate, polyvinyl butyrate acetate, vinyl acetate-maleic anhydride copolymer, A styrene-maleic acid monoester copolymer, a methyl acrylate-methacrylic acid copolymer, and a methacrylate-methacrylic acid-octyl acrylate copolymer. The method according to claim 1 or 3,
Wherein said pellet is present in a capsule or tablet.

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